Testing pulmonary physiology in ventilated non-human primates.

BACKGROUND: Animal models of respiratory viral infections are essential for investigating disease pathogenesis and the efficacy of antivirals and vaccine candidates. A major limitation in the research of respiratory diseases in animal models is correlating clinically relevant changes in pulmonary physiology with cellular and molecular mechanistic studies. Few animal models have captured and correlated physiologic changes in lung function and immune response within same experiment, which is critical given the heterogeneous nature of lung disease due to viral infections. In ventilated human patients, pulmonary physiology testing can be used to not only capture oxygenation, ventilation, but also pulmonary mechanics to yield quantitative measures of lung function and scalar tracings of flow-volume and pressure-volume loops. Application of this protocol during mechanical ventilation in non-human (NHP) models would represent a major advance in respiratory viral disease research. METHODS: We have applied and optimized a human pulmonary physiology testing protocol to ventilated pigtail macaques (Macaca nemestrina) at baseline and 5 days after influenza A (IAV) viral inoculation. RESULTS: The NHPs manifested clinical disease with hypothermia and loss of body weight. Declines in lung function were striking with a 66%-81% decline in P/F ratio, a measure of oxygenation reflecting the ratio of partial pressure of oxygen in arterial blood (PaO2 ) to the fraction of inspiratory oxygen concentration (FiO2 ). There was also a 16%-45% decline in lung compliance. CONCLUSION: We describe a new approach to performing pulmonary physiology testing protocol in non-human primates to better capture quantitative correlates of respiratory disease and demonstrate protection by therapeutics and vaccines.

Real-world epidemiology, treatment patterns and disease burden of patients diagnosed with chronic hepatitis B in Taiwan.

BACKGROUND AND AIMS: This study aimed to update the epidemiology, clinical, and economic outcomes of patients diagnosed with chronic hepatitis B (CHB) infection in Taiwan. METHODS: This is a retrospective observational study using claims data from the National Health Insurance Research Database. Cases were identified between 2010 and 2019 using CHB diagnosis codes and claims for alanine aminotransferase laboratory tests or CHB treatment within one year of the first CHB diagnosis. Patient characteristics, epidemiology, clinical, and economic outcomes were described. RESULTS: A total of 730 154 CHB-diagnosed cases were identified. The prevalence of diagnosed CHB increased from 1.13% in 2010 to 2.43% in 2019, with the highest occurring among those aged 55-64 years (4.76%) and 45-54 years (4.37%) and being higher in men (2.98%) than in women (2.21%). The majority of newly diagnosed CHB patients were 35 years of age or older (86.6%), with a median age of 49 years. After a median follow-up period of 6.42 years, 12.5%, 7.9%, 2.8%, and 0.35% were diagnosed with cirrhosis, decompensated cirrhosis, hepatocellular carcinoma, and liver transplantation respectively. Among 456 706 incident CHB-diagnosed patients, 17.4% had received at least one CHB medication, with the majority taking entecavir (67.9%). Patients with increasing disease severity had higher healthcare resource utilization, and inpatient costs accounted for 48.9%-65.5% of the overall medical cost in different health states. CONCLUSION: Despite the decreasing incidence of newly diagnosed CHB, the prevalence of diagnosed CHB remains high and poses a significant healthcare challenge owing to the high economic burden associated with the complications of CHB.

Impact of progesterone on innate immunity and cell death after influenza A virus H1N1 2009 infection of lung and placental cells in vitro.

The influenza A virus (IAV) 2009 H1N1 pandemic was associated with an increased risk of maternal mortality, preterm birth, and stillbirth. The underlying mechanism for severe maternal lung disease and stillbirth is incompletely understood, but IAV infection is known to activate innate immunity triggering the release of cytokines. Elucidating the impact of progesterone (P4), a key hormone elevated in pregnancy, on the innate immune and inflammatory response to IAV infection is a critical step in understanding the pathogenesis of adverse maternal-fetal outcomes. IAV H1N1 pdm/09 was used to infect cell lines Calu-3 (lung adenoma) and ACH-3P (extravillous trophoblast) with or without P4 (100 nM) at multiplicity of infections (MOI) 0, 0.5, and 3. Cells were harvested at 24 and 48 hours post infection (hpi) and analyzed for cytopathic effects (CPE), replicating virus (TCID50), cytotoxicity (Lactate Dehydrogenase (LDH) assay), and NLRP3 inflammasome activation (caspase-1 activity, fluorometric assay). Activation of antiviral innate immunity was quantified (RT-qPCR, Luminex) by measuring biomarker gene and protein expression of innate immune activation (IFIT1, IFNB), inflammation (IL6), interferon signaling (MXA), chemokines (IL-8, IL-10). Both Calu-3 and ACH-3P were highly permissible to IAV infection at each timepoint as demonstrated by CPE and recovery of replicating virus. In Calu-3, progesterone treatment was associated with a significant increase in cytotoxicity, increased gene expression of IL6, and increased protein expression of IFN-ß, IL-6, and IL-18. Conversely, in ACH-3P, progesterone treatment was associated with significantly suppressed cytotoxicity, decreased gene expression of IFNB, IL6 and IL1B, and increased protein expression of IFN-ß and IL-6. In both cell lines, caspase-1 activity was significantly decreased after progesterone treatment, indicating NLRP3 inflammasome activation was not underlying the higher cell death in Calu-3. In summary, these data provide evidence that progesterone plays a dual role by ameliorating viral infection in the placenta but exacerbating influenza A virus-associated injury in the lung through nongenomic modulation of the innate immune response.

Cervical-Vaginal Microbiome and Associated Cytokine Profiles in a Prospective Study of HPV 16 Acquisition, Persistence, and Clearance.

Persistent human papillomavirus (HPV) infections is necessary for the development of cervical cancers. Consequently, understanding the biologic mechanisms resulting in clearance is key in cancer prevention. Similar to other mucosal sites, it is expected that the local microbiome plays a significant role in shaping the immune response responsible for HPV clearance. Using cervical wash repository samples from a prospective study of HPV in women, this study investigates the microbiome and its associated inflammatory milieu during HPV 16 pre-acquisition, persistence and clearance states. For comparison, samples from women with no history of HPV ever during the study period were selected. We showed that 9 of 13 inflammatory cytokines were found to be significantly increased in the immediate post-clearance visit compared to the pre-acquisition or infection visits. Gardnerella vaginalis was associated with higher levels of inflammatory cytokines. Women with no history of HPV infection had similar cytokine profiles as those with HPV 16 post-clearance. This in vivo study documented an immune response shortly after HPV 16 clearance. G. vaginalis appeared to be involved in shaping this immune response. The appearance of G. vaginalis may have resulted from a shift from anti-microbial to anti-viral immune response with loss of bacterial control. The similar high levels of cytokines seen in women with no history of HPV suggest that a certain level of inflammatory surveillance is required to maintain an HPV negative state. This data may inform therapies such as probiotics or pro-inflammatory agents for treatment of persistent HPV.

Exercise plus caloric restriction lowers soluble RAGE in adults with chronic kidney disease.

INTRODUCTION: The incidence of chronic kidney disease (CKD) has increased in recent years. CKD is associated with obesity, type 2 diabetes, and cardiovascular disease, although the mechanism remains unclear. Elevated soluble form of the receptor for advanced glycation end products ( RAGE) is related to proinflammatory signaling pathways that may promote diabetic nephropathy and vascular dysfunction. Because lifestyle modification reduces systematic inflammation in adults with obesity and hyperglycaemia, the hypothesis that exercise plus caloric restriction would lower soluble RAGE in adults with CKD was tested in this study. METHODS: Eight adults (n = 6 females; age: 56.3 ± 2.8 y; BMI: 43.7 ± 2.2 kg/m2; 2-h OGTT glucose: 215 ± 9.8 mg/dL; eGFR: 49.6 ± 3.3 mL/min/1.73 m2) were enrolled in a 12-week pilot lifestyle intervention (supervised aerobic exercise [5 d/wk, up to 60 min/d at approximately 65%-85% HRmax] plus low-fat dietary counseling). Body composition (DXA), aerobic fitness (VO2max), insulin sensitivity (120 min 75 g OGTT; Matsuda Index), plasma levels of soluble RAGE and fetuin-A were measured before and after the intervention. RESULTS: Exercise reduced body weight, fasting glucose, and fetuin-A as well as increased VO2max, glucose tolerance, and insulin sensitivity (all P < .05). Lifestyle intervention decreased plasma soluble RAGE (pre: 1018.1 ± 163 vs post: 810.6 ± 119.6 ng/mL; P = .02), and the decrease was associated with a lower 2-hour blood glucose (r = 0.76, P = .03) and with increased insulin sensitivity (r = -0.90, P < .01). CONCLUSIONS: Exercise and caloric restriction are effective at lowering soluble RAGE in relation to glucose regulation in patients with CKD.

Gastric Bypass Surgery Improves the Skeletal Muscle Ceramide/S1P Ratio and Upregulates the AMPK/ SIRT1/ PGC-1α Pathway in Zucker Diabetic Fatty Rats.

PURPOSE: Roux-en-Y gastric bypass (RYGB) is associated with remission of type 2 diabetes. However, the cellular and molecular mechanisms remain unknown. We hypothesized that RYGB would increase peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC-1α), sirtuin-1 (SIRT1), AMPK/pAMPK, and citrate synthase (CS) protein expression and decrease insulin resistance and these changes would be mediated by sphingolipids, including ceramides and the sphingolipid metabolite sphingosine-1 phosphate (S1P). MATERIALS AND METHODS: Male ZDF rats were randomized to RYGB (n = 7) or sham surgery (n = 7) and harvested after 28 days. Total tissue ceramide, ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1), and S1P were quantified in the white gastrocnemius muscle using LC-ESI-MS/MS after separation with HPLC. Total SIRT1, AMPK, PGC-1α, and CS protein expression were measured by Western blot. RESULTS: Body weight, fasting glucose, insulin, and HOMA-IR decreased significantly after RYGB compared with sham control. These changes were paralleled by lower total ceramide (483.7 ± 32.3 vs. 280.1 ± 38.8 nmol/g wwt), C18:0 ceramide subspecies (P < 0.05), higher S1P (0.83 ± 0.05 vs. 1.54 ± 0.21 nmol/g wwt, P < 0.05), and a lower ceramide/S1P ratio (P < 0.05) in the RYGB versus sham group. AMPK, pAMPK, SIRT1, PGC-1α, and CS protein expression was also higher after RYGB (P < 0.05). The ceramide/S1P ratio correlated with weight loss (r = 0.48, P = 0.08), insulin resistance (r = 0.61, P = 0.02), PGC-1α (r = - 0.51, P < 0.06), CS (r = - 0.63, P = 0.01), and SIRT1 (r = - 0.54, P < 0.04). CONCLUSION: Our data demonstrate that sphingolipid balance, and increased AMPK, SIRT1, PGC-1α, and CS protein expression are part of the mechanism that contributes to the remission of diabetes after RYGB surgery.

Effect of Roux-en-Y gastric bypass on liver mitochondrial dynamics in a rat model of obesity.

Bariatric surgery provides significant and durable improvements in glycemic control and hepatic steatosis, but the underlying mechanisms that drive improvements in these metabolic parameters remain to be fully elucidated. Recently, alterations in mitochondrial morphology have shown a direct link to nutrient adaptations in obesity. Here, we evaluate the effects of Roux-en-Y gastric bypass (RYGB) surgery on markers of liver mitochondrial dynamics in a diet-induced obesity Sprague-Dawley (SD) rat model. Livers were harvested from adult male SD rats 90-days after either Sham or RYGB surgery and continuous high-fat feeding. We assessed expression of mitochondrial proteins involved in fusion, fission, mitochondrial autophagy (mitophagy) and biogenesis, as well as differences in citrate synthase activity and markers of oxidative stress. Gene expression for mitochondrial fusion genes, mitofusin 1 (Mfn1; P < 0.05), mitofusin 2 (Mfn2; P < 0.01), and optic atrophy 1 (OPA1; P < 0.05) increased following RYGB surgery. Biogenesis regulators, peroxisome proliferator-activated receptor gamma coactivator 1-alpha (PGC1α; P < 0.01) and nuclear respiratory factor 1 (Nrf1; P < 0.05), also increased in the RYGB group, as well as mitophagy marker, BCL-2 interacting protein 3 (Bnip3; P < 0.01). Protein expression for Mfn1 (P < 0.001), PGC1α (P < 0.05), BNIP3 (P < 0.0001), and mitochondrial complexes I-V (P < 0.01) was also increased by RYGB, and Mfn1 expression negatively correlated with body weight, insulin resistance, and fasting plasma insulin. In the RYGB group, citrate synthase activity was increased (P < 0.02) and reactive oxygen species (ROS) was decreased compared to the Sham control group (P < 0.05), although total antioxidant capacity was unchanged between groups. These data are the first to show an association between RYGB surgery and improved markers of liver mitochondrial dynamics. These observed improvements may be related to weight loss and reduced energetic demand on the liver, which could facilitate normalization of glucose homeostasis and protect against hepatic steatosis.

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Pancreatic Islets from Zucker Diabetic Fatty Rats.

PURPOSE: Diabetes and obesity are associated with inflammasome-mediated low-grade, chronic inflammation that may induce pancreatic beta-cell dysfunction and apoptosis. We examined the effects of Roux-en-Y gastric bypass (RYGB) surgery on NOD-like receptor family, pyrin domain containing-3 (NLRP3) inflammasome-related genes from pancreatic islets of Zucker diabetic fatty rats. MATERIALS AND METHODS: Islets were collected from Zucker diabetic fatty sham control and RYGB, 30 days after surgery. We assessed expression of genes that regulate glucose metabolism and the NLRP3 inflammasome (NLRP3, caspase-1, IL-1ß, IL-18, apoptosis-associated speck-like protein), IL-6, and monocyte chemoattractant protein-1. RESULTS: Gene expression for NLRP3 (p < 0.02), IL-1ß (p < 0.04), and IL-6 (p < 0.01) was reduced by RYGB and positively correlated with change in body weight. IL-1ß positively correlated with glucose AUC response. CONCLUSION: Suppression of the NLRP3 inflammasome in pancreatic islets may contribute to improved glycemic control after RYGB.

Effect of Roux-en-Y Gastric Bypass on the NLRP3 Inflammasome in Adipose Tissue from Obese Rats.

OBJECTIVE: Obesity is associated with low-grade chronic inflammation. We hypothesized that Roux-en-Y gastric bypass (RYGB) surgery would reduce activation of the NLRP3 inflammasome in metabolically active adipose tissue (AT) of obese rats, and this change would be related to decreases in body weight and improved glycemic control. METHODS: Omental, mesenteric and subcutaneous fat depots were collected from Sprague-Dawley rats: Sham control and RYGB; 90-days after surgery. NLRP3, caspase-1, apoptosis-associated speck-like protein (ASC), IL-1ß, IL-18, IL-6 and MCP-1 gene and protein expression were quantified. Glucose metabolism was assessed by oral glucose tolerance test (OGTT). RESULTS: Compared to Sham surgery controls, RYGB surgery decreased IL-6, MCP-1, NLRP3, IL-18, caspase-1 and ASC in omental fat, and decreased IL-6, MCP1, IL-1ß, IL-18, caspase-1 and ASC gene expression in mesenteric fat. We observed differential gene expression between visceral and subcutaneous fat for IL-6 and IL-1ß, both being downregulated by RYGB in visceral, and upregulated in subcutaneous depots. These changes in gene expression were accompanied by a decrease in NLRP3, ASC, IL-18, caspase-1 and IL-1ß protein expression in omental tissue. We found a positive correlation between caspase-1, ASC, MCP-1, IL-18 and IL-6 gene expression following surgery and glucose AUC response in omental fat, while the change in glucose AUC response correlated with caspase-1 gene expression in subcutaneous fat. CONCLUSION: This study demonstrates that bariatric surgery reverses inflammation in visceral adipose tissue by suppressing NLRP3 inflammasome activation. These are the first data to implicate the NLRP3 inflammasome in diabetes remission after RYGB surgery.

Improved insulin sensitivity after exercise training is linked to reduced plasma C14:0 ceramide in obesity and type 2 diabetes.

OBJECTIVE: To assess the effect of exercise training on insulin sensitivity and plasma ceramides in obesity and type 2 diabetes (T2D). METHODS: Twenty-four adults with obesity and normal glucose tolerance (NGT, n = 14) or diabetes (n = 10) were studied before and after a 12-week supervised exercise-training program (5 days/week, 1 h/day, 80-85% of maximum heart rate). Changes in body composition were assessed using hydrostatic weighing and computed tomography. Peripheral tissue insulin sensitivity was assessed by a 40 mU/m(2) /min hyperinsulinemic euglycemic clamp. Plasma ceramides (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1) were quantified using electrospray ionization tandem mass spectrometry after separation with HPLC. RESULTS: Plasma ceramides were similar for the subjects with obesity and NGT and the subjects with diabetes, despite differences in glucose tolerance. Exercise significantly reduced body weight and adiposity and increased peripheral insulin sensitivity in both groups (P < 0.05). In addition, plasma C14:0, C16:0, C18:1, and C24:0 ceramide levels were reduced in all subjects following the intervention (P < 0.05). Decreases in total (r = -0.51, P = 0.02) and C14:0 (r = -0.56, P = 0.009) ceramide were negatively correlated with the increase in insulin sensitivity. CONCLUSIONS: Ceramides are linked to exercise training-induced improvements in insulin sensitivity, and plasma C14:0 ceramide may provide a specific target for investigating lipid-related insulin resistance in obesity and T2D.

Exercise-induced lowering of fetuin-A may increase hepatic insulin sensitivity.

INTRODUCTION: Fetuin-A is a novel hepatokine, and there is preliminary evidence that it may contribute to the pathogenesis of type 2 diabetes. Exercise reduces fetuin-A, but the specific metabolic effects particularly as they relate to the regulation of insulin resistance are unknown. This led us to examine the effect of exercise training on circulating fetuin-A in relation to skeletal muscle and/or hepatic insulin resistance in obese adults. METHODS: Twenty older adults (66.3 ± 0.9 yr; body mass index, 34.1 ± 1.2 kg · m(-1)) participated in this prospective 12-wk study and underwent supervised exercise training (5 d · wk(-1), 60 min · d(-1) at approximately 85% HRmax). Insulin resistance was assessed using the euglycemic-hyperinsulinemic clamp (40 mU · m(-2) · min(-1)) with isotope dilution ([6,6-H2]-glucose). Skeletal muscle insulin sensitivity (rate of glucose disposal), hepatic insulin resistance (rate of glucose appearance × fasting insulin), metabolic flexibility (respiratory quotient clamp - respiratory quotient fasting), fetuin-A, high-molecular weight adiponectin, high-sensitivity C-reactive protein, leptin, and body fat (dual energy x-ray absorptiometry) were measured before and after the intervention. RESULTS: Exercise reduced body fat, high-sensitivity C-reactive protein, leptin and hepatic as well as skeletal muscle insulin resistance (each, P < 0.05). Fetuin-A was decreased by approximately 8% (pre, 1.01 ± 0.08, vs post, 0.89 ± 0.06 g · L(-1); P < 0.05) after the intervention, and lower fetuin-A after exercise correlated with lower hepatic insulin resistance (r = -0.46, P < 0.01), increased metabolic flexibility (r = -0.70, P < 0.01) and high-molecular weight adiponectin (r = -0.57, P < 0.01). CONCLUSIONS: Fetuin-A may contribute to exercise training-induced improvements in hepatic insulin resistance, CHO utilization, and inflammation in older obese adults. Further work is required to determine the cellular mechanism(s) of action for fetuin-A because this hepatokine is related to type 2 diabetes risk.

Fetuin-A is linked to improved glucose tolerance after short-term exercise training in nonalcoholic fatty liver disease.

Fetuin-A is synthesized in the liver and may be associated with nonalcoholic fatty liver disease (NAFLD) and type 2 diabetes. Lifestyle-induced weight loss reduces fetuin-A, but the effect of exercise alone is unknown. We determined the effect of short-term exercise training on plasma fetuin-A in 13 (50.5 ± 3.4 yr) obese adults (body mass index, 33.3 ± 0.9 kg/m(2)) with clinically diagnosed NAFLD. Subjects participated in 7 days of supervised exercise training (60 min/day at ∼85% maximum heart rate) and were instructed to maintain their normal caloric and macronutrient intake. Insulin resistance was assessed by an oral glucose tolerance test. Hepatic triglyceride content (HTGC) was determined by proton MRI. We used C2C12 skeletal muscle cells to examine the direct effect of fetuin-A on 2-deoxyglucose uptake, insulin signaling [phosphorylation of Akt and AS160 (pAkt and pAS160, respectively)], and glucose transporter-4 (GLUT-4) translocation. Insulin resistance was reduced by 29% (P < 0.05), and glucose area under the curve (AUC) was decreased by 13% (P < 0.01) after the 7 days of exercise. Furthermore, circulating fetuin-A was decreased by 11% (4.2 ± 03 vs. 3.6 ± 0.2 nM; P < 0.02), and this change correlated with reduced insulin resistance (r = 0.62; P < 0.04) and glucose AUC (r = 0.58; P < 0.04). Importantly, the exercise program did not change body weight (P = 0.12), HTGC (P = 0.73), or aerobic capacity (P = 0.14). In vitro experiments revealed that fetuin-A decreased skeletal muscle glucose uptake by downregulating pAkt and pAS160 and subsequent GLUT-4 translocation to the plasma membrane. Together, our findings highlight a role for fetuin-A in skeletal muscle insulin resistance and suggest that part of the exercise-induced improvement in glucose tolerance in patients with NAFLD may be due to lowering fetuin-A.

Lower dipeptidyl peptidase-4 following exercise training plus weight loss is related to increased insulin sensitivity in adults with metabolic syndrome.

Dipeptidyl peptidase-4 (DPP-4) is a circulating glycoprotein that impairs insulin-stimulated glucose uptake and is linked to obesity and metabolic syndrome. However, the effect of exercise on plasma DPP-4 in adults with metabolic syndrome is unknown. Therefore, we determined the effect of exercise on DPP-4 and its role in explaining exercise-induced improvements in insulin sensitivity. Fourteen obese adults (67.9±1.2 years, BMI: 34.2±1.1kg/m(2)) with metabolic syndrome (ATP III criteria) underwent a 12-week supervised exercise intervention (60min/day for 5 days/week at ∼85% HRmax). Plasma DPP-4 was analyzed using an enzyme-linked immunosorbent assay. Insulin sensitivity was measured using the euglycemic-hyperinsulinemic clamp (40mU/m(2)/min) and estimated by HOMA-IR. Visceral fat (computerized tomography), 2-h glucose levels (75g oral glucose tolerance), and basal fat oxidation as well as aerobic fitness (indirect calorimetry) were also determined before and after exercise. The intervention reduced visceral fat, lowered blood pressure, glucose and lipids, and increased aerobic fitness (P<0.05). Exercise improved clamp-derived insulin sensitivity by 75% (P<0.001) and decreased HOMA-IR by 15% (P<0.05). Training decreased plasma DPP-4 by 10% (421.8±30.1 vs. 378.3±32.5ng/ml; P<0.04), and the decrease in DPP-4 was associated with clamp-derived insulin sensitivity (r=-0.59; P<0.04), HOMA-IR (r=0.59; P<0.04) and fat oxidation (r=-0.54; P<0.05). Increased fat oxidation also correlated with lower 2-h glucose levels (r=-0.64; P<0.02). Exercise training reduces plasma DPP-4, which may be linked to elevated insulin sensitivity and fat oxidation. Maintaining low plasma DPP-4 concentrations is a potential mechanism whereby exercise plus weight loss prevents/delays the onset of type 2 diabetes in adults with metabolic syndrome.

Reduced cardiovascular risk after bariatric surgery is linked to plasma ceramides, apolipoprotein-B100, and ApoB100/A1 ratio.

BACKGROUND: Obesity-associated hyperlipidemia and hyperlipoproteinemia are risk factors for cardiovascular disease (CVD). Recently, ceramide-derived sphingolipids were identified as a novel independent CVD risk factor. We hypothesized that the beneficial effect of Roux-en-Y gastric bypass (RYGB) on CVD risk is related to ceramide-mediated improvement in lipoprotein profile. METHODS: A prospective study of patients undergoing RYGB was conducted. The patients' clinical data and biochemical markers related to cardiovascular risk were documented. Plasma ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1), apolipoprotein (Apo)B100 and ApoA1 were quantified preoperatively and 3 and 6 months after RYGB, as was the Framingham risk score. Brachial artery reactivity testing was performed before and 6 months after RYGB. RESULTS: Ten patients (9 women; age 48.6 ± 9.6 yr; body mass index, 48.5 ± 5.8 kg/m(2)) were included in the present study. At 6 months postoperatively, the mean body mass index had decreased to 35.7 ± 5.0 kg/m(2), corresponding to 51.3% ± 10.0% excess weight loss. The fasting total cholesterol, triglycerides, low-density lipoprotein, free fatty acids, ApoB100, ApoB100/ApoA1 ratio and insulin resistance estimated from Homeostasis Model of Assessment of Insulin Resistance were significantly reduced compared with the preoperative values. The ApoB100/ApoA1 ratio correlated with a reduction in ceramide subspecies (C18:0, C18:1, C20:0, C24:0, and C24:1; P < .05). ApoB100 and the ApoB100/ApoA1 ratio also correlated positively with the reduction in triglycerides, low-density lipoprotein, and Homeostasis Model of Assessment of Insulin Resistance (P < .05). Brachial artery reactivity testing correlated inversely with ApoB100 and total ceramide (P = .05). Furthermore, the change in brachial artery reactivity testing correlated with the decrease in C16:0 (P < .03). CONCLUSION: Our data suggest that improvements in lipid profiles and CVD risk factors after gastric bypass surgery could be linked to changes in ceramide lipids. Mechanistic studies are needed to determine whether this link is causative or purely correlative.

Gastric bypass surgery reduces plasma ceramide subspecies and improves insulin sensitivity in severely obese patients.

Bariatric surgery is associated with near immediate remission of type 2 diabetes and hyperlipidemia. The mechanisms underlying restoration of normal glucose tolerance postoperatively are poorly understood. Herein, we examined the effect of Roux-en-Y gastric bypass surgery (RYGB) on weight loss, insulin sensitivity, plasma ceramides, proinflammatory markers, and cardiovascular risk factors before and at 3 and 6 months after surgery. Thirteen patients (10 female; age 48.5 ± 2.7 years; BMI, 47.4 ± 1.5 kg/m(2)) were included in the study, all of whom had undergone laparoscopic RYGB surgery. Insulin sensitivity, inflammatory mediators and fasting lipid profiles were measured at baseline, 3 and 6 months postoperatively, using enzymatic analysis. Plasma ceramide subspecies (C14:0, C16:0, C18:0, C18:1, C20:0, C24:0, and C24:1) were quantified using electrospray ionization tandem mass spectrometry after separation with HPLC. At 3 months postsurgery, body weight was reduced by 25%, fasting total cholesterol, triglycerides, low-density lipoproteins, and free fatty acids were decreased, and insulin sensitivity was increased compared to presurgery values. These changes were all sustained at 6 months. In addition, total plasma ceramide levels decreased significantly postoperatively (9.3 ± 0.5 nmol/ml at baseline vs. 7.6 ± 0.4 at 3 months, and 7.3 ± 0.3 at 6 months, P < 0.05). At 6 months, the improvement in insulin sensitivity correlated with the change in total ceramide levels (r = -0.68, P = 0.02), and with plasma tumor necrosis factor-α (TNF-α) (r = -0.62, P = 0.04). We conclude that there is a potential role for ceramide lipids as mediators of the proinflammatory state and improved insulin sensitivity after gastric bypass surgery.

Early effects of gastric bypass on endothelial function, inflammation, and cardiovascular risk in obese patients.

BACKGROUND: Obesity is associated with a chronic low-grade inflammatory state, insulin resistance, and endothelial dysfunction, all of which contribute to increased risk of cardiovascular disease. We hypothesized that gastric bypass would produce rapid improvements in endothelial function, reduce inflammation, and lead to a decrease in cardiovascular risk. METHODS: We performed a prospective study of morbidly obese patients who underwent laparoscopic Roux-en-Y gastric bypass (RYGB). Clinical data, biochemical markers of inflammation, and parameters indicative of cardiovascular risk were collected preoperatively and at 3 and 6 months postoperatively. Metabolic and inflammatory mediators that were quantified included C-reactive protein, fibrinogen, PAI-1, IL-6, IL-10, IL-1Ra, adiponectin, leptin, triglycerides, total cholesterol, HDL, LDL, glucose, insulin, and HbA1c. Brachial artery reactivity testing (BART) was performed to assess peripheral arterial endothelial function, and Framingham cardiovascular risk score (FRS) was calculated on all study participants pre- and postoperatively. RESULTS: Fifteen patients (11 female) were enrolled (age = 49.2 ± 10.4 years; BMI = 48.1 ± 5.3 kg/m(2)). Six months post RYGB, mean BMI decreased to 35.4 ± 4.5, corresponding to 51.7% excess weight loss (P < 0.001). Mean waist circumference decreased significantly from 132 cm at baseline to 110 cm at 3 months (P = 0.003) and 107 cm at 6 months (P < 0.001). Six months after RYGB, weight loss led to significant improvements in clinical parameters indicative of cardiovascular disease or risk, including brachial artery diameter, endothelial independent vasodilation, and FRS. Favorable improvements in the proinflammatory markers CRP (P = 0.01) and leptin (P = 0.005), the anti-inflammatory mediator adiponectin (P = 0.002), and insulin sensitivity (HOMA-IR, P = 0.007) were evident at 3 months. At 6 months, improvements in CRP, leptin, and fasting insulin were maintained and fibrinogen levels also decreased (P = 0.047). Adiponectin continued to increase at 6 months (P = 0.004). CONCLUSION: Gastric bypass is associated with early reversal of endothelial dysfunction, a more favorable inflammatory milieu, and, most importantly, a reduction in cardiovascular risk.

Pancreatic islet isolation after gastric bypass in a rat model: technique and initial results for a promising research tool.

BACKGROUND: Roux-en-Y gastric bypass (RYGB) affords a high remission rate of type 2 diabetes mellitus among morbidly obese diabetic patients. We report the use of the isolated islet technique to assess pancreatic function and glucoregulatory mechanisms after RYGB surgery. METHODS: A total of 15 adult, male, Sprague Dawley diet-induced obese rats were randomly divided into 3 experimental groups: sham, RYGB, and pair-fed, with 5 rats in each group. The body weight was measured at baseline and every week for 4 weeks. Pancreatic islet function was assessed in vitro according to the amount of insulin secreted from isolated islets incubated in 2 mM and 20 mM glucose for 1 hour at 37 °C. Fasting plasma glucose, insulin, glucagon-like peptide-1, PYY3-36, and glucose-dependent insulinotropic peptide were measured at baseline and 28 days after surgery. RESULTS: The baseline body weight was 917 ± 61, 831 ± 42, and 927 ± 43 g for the sham, RYGB, and pair-fed groups, respectively. The RYGB group lost 32% body weight compared with 16% for the sham and 24% for the pair-fed groups. Glucose-stimulated insulin secretion from the isolated islets in the RYGB group was greater than in the comparison groups (P = .04) at 4 weeks after surgery. Fasting plasma glucagon-like peptide-1 and PYY3-36 were significantly increased at 4 weeks in the RYGB group. CONCLUSION: Islet isolation and stimulation in the present animal model was feasible, affords a direct measurement of pancreatic islet function, and might provide a useful tool to study the effects of RYGB on pancreatic function and the relationship between islet cell function and incretin production after bariatric surgery.

Quantification of ceramide species in biological samples by liquid chromatography electrospray ionization tandem mass spectrometry.

We present an optimized and validated liquid chromatography electrospray ionization tandem mass spectrometry (LC-ESI-MS/MS) method for the simultaneous measurement of concentrations of different ceramide species in biological samples. The method of analysis of tissue samples is based on Bligh and Dyer extraction, reverse-phase high-performance liquid chromatography separation, and multiple reaction monitoring of ceramides. Preparation of plasma samples also requires isolation of sphingolipids by silica gel column chromatography prior to LC-ESI-MS/MS analysis. The limits of quantification were in a range of 0.01-0.50ng/ml for distinct ceramides. The method was reliable for inter- and intraassay precision, accuracy, and linearity. Recoveries of ceramide subspecies from human plasma, rat liver, and muscle tissue were 78 to 91%, 70 to 99%, and 71 to 95%, respectively. The separation and quantification of several endogenous long-chain and very-long-chain ceramides using two nonphysiological odd chain ceramide (C17 and C25) internal standards was achieved within a single 21-min chromatographic run. The technique was applied to quantify distinct ceramide species in different rat tissues (muscle, liver, and heart) and in human plasma. Using this analytical technique, we demonstrated that a clinical exercise training intervention reduces the levels of ceramides in plasma of obese adults. This technique could be extended for quantification of other ceramides and sphingolipids with no significant modification.

Acute altitude-induced hypoxia suppresses plasma glucose and leptin in healthy humans.

To examine the effects of acute altitude-induced hypoxia on the hormonal and metabolic response to ingested glucose, 8 young, healthy subjects (5 men and 3 women; age, 26 +/- 2 years; body mass index, 23.1 +/- 1.0 kg/m(2)) performed 2 randomized trials in a hypobaric chamber where a 75-g glucose solution was ingested under simulated altitude (ALT, 4300 m) or ambient (AMB, 362 m) conditions. Plasma glucose, insulin, C-peptide, epinephrine, leptin, and lactate concentrations were measured at baseline and 30, 60, 90, and 120 minutes after glucose ingestion during both trials. Compared with AMB, the plasma glucose response to glucose ingestion was reduced during the ALT trial (P = .04). There were no differences in the insulin and C-peptide responses between trials or in insulin sensitivity based on the homeostasis model assessment of insulin resistance. Epinephrine and lactate were both elevated during the ALT trial (P < .05), whereas the plasma leptin response was reduced compared with AMB (P < .05). The data suggest that the plasma glucose response is suppressed at ALT, but this is not due to insulin per se because insulin and C-peptide levels were similar for both trials. Elevated plasma epinephrine and lactate during ALT are indicative of increased glycogenolysis, which may have masked the magnitude of the reduced glucose response. We conclude that, during acute altitude exposure, there is a rapid metabolic response that is accompanied by a shift in the hormonal milieu that appears to favor increased glucose utilization.

Dipropionylcysteine ethyl ester compensates for loss of citric acid cycle intermediates during post ischemia reperfusion in the pig heart.

PURPOSE: During reperfusion, following myocardial ischemia, uncompensated loss of citric acid cycle (CAC) intermediates may impair CAC flux and energy transduction. Propionate has an anaplerotic effect when converted to the CAC intermediate succinyl-CoA, and may improve contractile recovery during reperfusion. Antioxidant therapy with N-acetylcysteine decreases reperfusion injury. To synergize the antioxidant effects of cysteine with the anaplerotic effects of propionate, we synthesized a novel bi-functional compound, N,S-dipropionyl cysteine ethyl ester (DPNCE) and tested its anaplerotic and anti-oxidative capacity in anesthetized pigs. METHODS: Ischemia was induced by a 70% reduction in left anterior descending coronary artery flow for one hour, followed by 1 h of reperfusion. After 30 min of ischemia and throughout reperfusion animals were treated with saline or intravenous DPNCE (1.5 mg x kg(-1) x min(-1), n = 8/group). Arterial concentrations and myocardial propionate, cysteine, free fatty acids, glucose and lactate uptakes, cardiac mechanical functions, myocardial content of CAC intermediates and oxidative stress were assessed. RESULTS: Ischemia resulted in reduction in myocardial tissue concentration of CAC intermediates. DPNCE treatment elevated arterial propionate and cysteine concentrations and myocardial propionate uptake, and increased myocardial concentrations of citrate, succinate, fumarate, and malate compared to saline treated animals. DPNCE treatment did not affect blood pressure or myocardial contractile function, but increased arterial free fatty acid concentration and myocardial fatty acid uptake. Arterial cysteine concentration was elevated by DPNCE, but there was negligible myocardial cysteine uptake, and no change in markers of oxidative stress. CONCLUSION: DPNCE elevated arterial cysteine and propionate, and increased myocardial concentration of CAC intermediates, but did not affect mechanical function or oxidative stress.